National Research Council - Institute of Biomedical Technology

Dr. Gianluca De Bellis

Genomics, Transcriptomics & Epigenomics

in Melanoma

National Research Council - Institute of Biomedical Technology

Genomics, Transcriptomics & Epigenomics in Melanoma

15 Melanomas

2 Melanocites

Exome Seq

RNA-seq

Methylation

miRNA

mRNA

SNParray

Proteomics

National Research Council - Institute of Biomedical Technology

Genomics

15 Melanomas

2 Melanocites

Exome Seq

National Research Council - Institute of Biomedical Technology

NIMBLEGEN 2.1 ARRAY AGILENT SURE SELECT

Solid array Solution

Fragment length ~500bp Fragment length ~300bp

20-mer Adaptors (both ends), BAD with ILLUMINA

No Adaptors

~35 Mb exon target ~50 Mb exon target

National Research Council - Institute of Biomedical Technology

Nimblegen 2.1 Agilent SureSelect

Sample ReadsCapture

CoverageDepth Reads

Capture Coverage

Depth

Me01 19,447,744 24,54% 22,5 X 69,721,034 94,98% 44,54 X

Me04 15,273,918 60,39% 11,51 X 84,459,524 94,45% 50,78 X

Me05 17,1785,980 91,40% 9,79 X 50,671,560 94,66% 32,71 X

Comparison Nimblegen vs Agilent SureSelect

National Research Council - Institute of Biomedical Technology

Raw Reads

Mapping

LocalRealignment

Duplicate Marking

Base QualityRecalibration

Input

Output

GATKSNP Call

SNVINDEL

Raw Variants

RawSNV

RawINDEL

External Data

KnownSNP

KnownGenotypes

Analysis-readyVariants

Novel SNV

SIFTPrediction

Quality Filtering

VariantAnnotation

High QualityCodingVariant

Database

IntegrativeAnalysis

withTranscriptome

MAQ

SAMTools

National Research Council - Institute of Biomedical Technology

Exome

Overall variants RS non RSCoding non RS

nonsynonimous DAMAGING

37.469 19.088 18.381 3.771 228

66.921 59.611 7.310 4.975 117

106.046 79.341 26.705 6.951 423

65.504 52.711 12.793 3.646 146

62.282 54.956 7.326 4.667 72

67.231 59.975 7.256 5.242 131

86.663 75.578 11.085 5.859 143

59.797 49.942 9.855 4.231 100

68.062 59.002 9.060 4.522 150

69.044 61.167 7.877 5.331 335

149.891 130.878 19.013 6.425 129

61.619 54.287 7.332 4.921 180

174.135 158.630 15.505 6.518 118

139.075 124.170 14.905 6.506 92

204.189 170.174 34.015 8.311 214

147.893 124.928 22.965 9.174 104

1.565.821 1.334.438 231.383 91.050

85% ON AVERAGE 5% ON AVERAGE

National Research Council - Institute of Biomedical Technology

National Research Council - Institute of Biomedical Technology

National Research Council - Institute of Biomedical Technology

National Research Council - Institute of Biomedical Technology

Genomics

National Research Council - Institute of Biomedical Technology

Transcriptomics

15 Melanomas

2 Melanocites

RNA-seq

Sample Basi coperte 8X EsomaBasi coperte 8X

Trascrittoma

Me01 4.364.942 34.441.297

Me02 12.620.462 30.403.923

Me03 13.606.281 34.178.638

Me04 7.833.813 38.474.628

Me05 11.460.861 33.910.183

Me06 15.159.817 21.429.893

Me07 16.902.935 30.347.551

Me08 8.760.350 33.337.895

Me09 14.555.472 34.125.087

Me10 14.546.849 31.873.272

Me11 21.839.058 33.072.609

Me12 12.514.867 31.027.082

Me13 23.804.340 34.227.522

Me14 24.439.547 37.490.114

Me15 25.530.726 32.864.914

Melanociti01 24.793.849 32.130.548

Perfect

Match

(total

overlap)

Contained

(partial

overlap)

Novel Isoform

(at least one

transcript

with a splice

variant)

Generic

exonic

overlap

(exon

intron

overlap

<10bp or no

exon-intron

junction

match)

Single Ref

exon-intron

overlap

(>10bp) Pre

mRNA

Fragment

Intronic

transcripts

Unknown

intergenic

transcript

s

Mapping

errors,

polymerase

errors

Me01 7761 5717 29276 (Total)

7220 (10X

mean

coverage)

1538 1117 10330 19567 3587

Me02 6886 8032 21687 (Tot)

5926 (10X)

1784 1862 17754 15567 3037

Me03 7208 4688 36512 (Tot)

8776 (10X)

1556 1195 11078 13970 3663

Me04 7882 5207 44593 (Tot)

11772 (10X)

1760 1175 10165 17691 4302

Me05 7487 5500 33102 (Tot)

7623 (10X)

1168 1531 8850 12172 3130

Me06 4955 12021 15347 (Tot)

4310 (10X)

1875 1574 15630 12067 2603

Me07 6625 7699 22128 (Tot)

5591 (10X)

1595 1546 14348 10455 2705

Me08 7849 4885 32328 (Tot)

6932 (10X)

1307 1036 8057 10080 2873

Me09 7610 4641 36712 (Tot)

8099 (10X)

928 1309 6592 9425 3119

Transcripts with perfect match

Transcripts with perfect match

Novel Isoform (alternative splicing)

Potential novel isoform if exist at least one transcript with a splice junction variant

Pre mRNA Fragment

Possible pre mRNA fragment (single exon tran script overlapping a reference exon and at least 10bp of a reference intron)

Intronic transcripts

Transcript falling entirely within a reference intron

Unknown intergenic transcripts

NOVEL ISOFORM DIFF. EXPRESSED IN MELANOMA VS MELANOCITA

MELANOMA

NOVEL ISOFORM DIFF. EXPRESSED IN MELANOMA VS MELANOCITA

MELANOCITA

National Research Council - Institute of Biomedical Technology

COMBINING EXOME AND

TRANSCRIPTOME DATA

National Research Council - Institute of Biomedical Technology

National Research Council - Institute of Biomedical Technology

National Research Council - Institute of Biomedical Technology

BRAF - V600E status in Ex & Tx

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mel1Mel2

Ex ND NEG NEG ND ND POS NEG ND POS NEG POS POS POS POS NEG NEG /

Tx POS NEG NEG POS NEG ND ND POS POS NEG POS POS POS POS NEG NEG NEG

+ - - + - + - + + - + + + + - - -

Microbial GenomicsTargeted ResequencingCMTBrugadaCardiomiopatie

Whole transcriptomeSequencing & miRNA

Structural genomics& Epigenomics

Ancient DNA

National Research Council - Institute of Biomedical Technology

Acknowledgements

• Ermanno Rizzi• Roberta Bordoni• Clelia Peano• Ingrid Cifola• Clarissa Consolandi• Valentina Tinaglia• Marica Proverbio• Cristina Battaglia

• Giorgio Corti• Marco Severgnini• Fabio Fuligni• Alessandro Pietrelli• Eleonora Mangano• Fabio Frascati

Molecular

Biology

Bioinformatics

Tech Support

Giancarlo Caramenti

Giada Caredda

Maria Vurchio

Dr. Vincenzo Russo

Terapia genica dei tumori

HSR - Milan